Chlorophytum comosum L. plants are known to effectively absorb air pollutants, including formaldehyde (HCHO). Since the metabolic and defense responses of C. comosum to HCHO are poorly understood, in the present study, biochemical changes in C. comosum leaves induced by 48 h exposure to exogenous HCHO, applied as 20 mg m−3, were analyzed. The observed changes showed that HCHO treatment caused no visible harmful effects on C. comosum leaves and seemed to be effectively metabolized by this plant. HCHO application caused no changes in total chlorophyll (Chl) and Chl a content, increased Chl a/b ratio, and decreased Chl b and carotenoid content. HCHO treatment affected sugar metabolism, towards the utilization of sucrose and synthesis or accumulation of glucose, and decreased activities of aspartate and alanine aminotransferases, suggesting that these enzymes do not play any pivotal role in amino acid transformations during HCHO assimilation. The total phenolic content in leaf tissues did not change in comparison to the untreated plants. The obtained results suggest that HCHO affects nitrogen and carbohydrate metabolism, effectively influencing photosynthesis, shortly after plant exposure to this volatile compound. It may be suggested that the observed changes are related to early HCHO stress symptoms or an early step of the adaptation of cells to HCHO treatment. The presented results confirm for the first time the direct influence of short time HCHO exposure on the studied parameters in the C. comosum plant leaf tissues.